Open AccessOptimization of Taq DNA polymerase enzyme expression in Escherichia coli
Author(s) -
Fateme Moazen,
Ali Rastegari,
Sayed Mehdi Hoseini,
Mojtaba Panjehpour,
Mehran Miroliaei,
Hamid Mir Mohammad Sadeghi
Publication year - 2012
Publication title -
advanced biomedical research
Language(s) - English
Resource type - Journals
ISSN - 2277-9175
DOI - 10.4103/2277-9175.103004
Subject(s) - microbiology and biotechnology , recombinant dna , polymerase , escherichia coli , taq polymerase , polymerase chain reaction , lac operon , restriction enzyme , dna polymerase , hindiii , dna , plasmid , chemistry , biology , gene , thermus aquaticus , biochemistry
Background: In the present study, we optimized the experimental conditions using pET15b expression vector to obtain large amounts of Taq DNA polymerase.Materials and Methods: Correct framing of the gene in the expression vector pET15b and its orientation were analyzed by digestion and sequencing. Production of Taq DNA polymerase in Escherichia coli BL21 (DE3) cells was induced by incubation with different concentrations of IPTG. Optimum production occurred with the addition of 1mM IPTG for 2h. The activity of the obtained enzyme was measured by comparing the intensities of the produced DNA bands in PCR reactions.Results: Recombinant plasmid containing taq polymerase gene was confirmed by restriction digestion and DNA sequencing. Purified protein was identified by Western blotting. Optimum condition for the production of the enzyme was induction with 1mM IPTG for 23h. Addition of NP40 increased enzyme stability.Conclusion: We expressed the recombinant Taq DNA polymerase in E. coli using a T7based promoter system and obtained an active and stable enzyme